clinical study phase i study of anti-cd3 x anti-her2...

Clinical StudyPhase I Study of Anti-CD3 x Anti-Her2 Bispecific Antibody inMetastatic Castrate Resistant Prostate Cancer Patients

Ulka Vaishampayan,1,2 Archana Thakur,1 Ritesh Rathore,3

Nicola Kouttab,4 and Lawrence G. Lum1,2,5

1Department of Oncology, Wayne State University and Karmanos Cancer Institute, Detroit, MI 48201, USA2Department of Medicine, Wayne State University, Detroit, MI 48201, USA3Roger Williams Medical Center, Providence, RI 02908, USA4Department of Pathology, Roger Williams Medical Center, Providence, RI 02908, USA5Department of Immunology and Microbiology, Wayne State University, Detroit, MI 48201, USA

Correspondence should be addressed to Lawrence G. Lum; [email protected]

Received 10 November 2014; Revised 9 January 2015; Accepted 15 January 2015

Academic Editor: Hendrik Van Poppel

Copyright © 2015 Ulka Vaishampayan et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Background. New nontoxic targeted approaches are needed for patients with castrate resistant prostate cancer (CRPC). Ourpreclinical studies show that activated T cells (ATC) armed with anti-CD3 x anti-Her2 bispecific antibody (Her2Bi) kill prostatecancer cells lines, induce aTh

1cytokine pattern upon engagement of tumor cells, prevent the development of prostate tumors, and

retard tumor growth in immunodeficient mice. These studies provided strong rationale for our phase I dose-escalation pilot studyto test ATC armed with Her2Bi (aATC) for safety in men with CRPC. Methods. Seven of 8 men with CRPC were evaluable afterreceiving two infusions per week for 4 weeks. The men received 2.5, 5 or 10 × 109 aATC per infusion with low dose interleukin-2 and granulocyte-macrophage colony stimulating factor. Results. There were no dose limiting toxicities, and there was 1 partialresponder and 3 of 7 patients had significant decreases in their PSA levels and pain scores. Immune evaluations of peripheralblood mononuclear cells in 2 patients before and after immunotherapy showed increases in IFN-𝛾 EliSpot responses and Th

1

serum cytokines. Conclusions. These results provide a strong rationale for developing phase II trials to determine whether aATCare effective for treating CRPC.

1. Background

In 2010, the estimatedmortality related to prostate cancer wasnoted to be increasing. With increasing numbers of prostatecancer patients with prostate specific antigen (PSA) relapse,the populationwithmetastatic disease is expected to increase.Although androgen deprivation therapy (ADT) is initiallyvery effective in patients, the disease eventually progressesinto castrate resistant prostate cancer (CRPC). Failure ofADTis inevitable with disease progression associated with risingPSA levels, clinical symptoms, or abnormal scans. Despitetreatment with docetaxel-based chemotherapy, patients withmetastatic CRPC have an expected median survival of 18–20 months. Although targeted therapies such as abirateroneand MDV-3100 may improve overall survival, complete

remissions are unlikely and patients eventually progress withpain, neuropathic compression, and fractures. Immunother-apy is an attractive nontoxic clinical strategy for patientswith CRPC with the possibility of inducing prolonged remis-sions. Dendritic cell vaccine, Sipuleucel-T, has been shownto improve overall survival in patients with CRPC [1]. In2010, Sipuleucel-T was approved by the US Food and DrugAdministration (FDA) for patients with metastatic CRPC asan approved therapeutic option. The approval of Sipuleucel-T sets the stage for other cell-based immunotherapies forprostate cancer.

Her2/neu (Her2) overexpression as tested by immunohis-tochemistry (IHC) is reported in 42–70% of prostate cancers[2, 3]. Overexpression of Her2 appears to be an unfavorableprognostic factor and is associated with lower survival and

Hindawi Publishing CorporationProstate CancerVolume 2015, Article ID 285193, 10 pageshttp://dx.doi.org/10.1155/2015/285193

2 Prostate Cancer

higher relapse rates than those of Her2 negative prostate can-cers [2]. Furthermore, serum Her2 levels were significantlyhigher in prostate cancer patients than in control subjectswithout cancer and serum Her2 levels were significantlyhigher in patients with metastatic disease than those withoutmetastatic disease [4]. Likewise, in patients with metastaticdisease, patients with higher serum Her2 levels had a shortertime to recurrence when compared to those with lowerHer2 levels [4]. Her2 overexpression in prostate cancercells also increased with progression to androgen inde-pendence [5, 6]. Forced Her2 overexpression in a LAPC-4prostate cancermodel confers androgen-independent growthto androgen-dependent prostate cancer cells [7]. These stud-ies provide insights into the clinical role of Her2 expres-sion in clinical progression and survival of patients withCRPC.

Several phase II clinical trials involved targeting Her2prostate cancers by infusing trastuzumab (Herceptin) intoprostate cancer patients; however, one study closed due toinability to accrue sufficient numbers of HER2+ patients [8]and another study failed to demonstrate benefit in CRPCpatients [9].The advantage of using anti-CD3 activatedT cells(ATC) redirected by bispecific antibodies (BiAbs) over usingmonoclonal antibodies or BiAbs alone is that the armingof ATC with BiAbs combines independent mechanisms ofcytotoxicity into a single biologic drug. Arming ATC withanti-CD3 x anti-Her2 BiAb (Her2Bi) targets T cells to Her2on the tumor cells. Arming with Her2Bi transforms everyATC into a specific cytotoxic T cell directed at both highand lowHer2 expressing targets. Our preclinical studies showthat ATC armed with Her2Bi exhibited high levels of non-MHC restricted cytotoxicity directed at PC-3, DU-145, andLNCaP prostate cancer cell lines and produced tumoricidalcytokines such as interferon 𝛾 (IFN𝛾), tumor necrosis factor𝛼 (TNF𝛼), and GM-CSF as well as MIP1alpha and RANTES[10–22]. Furthermore, Her2Bi-armed ATC coinjected withPC-3 localized to PC-3 xenografts and prevented growth oftumors or induced remissions when injected intratumorallyin severe combined immunodeficient beige mice [23]. Toevaluate this strategy, we performed a phase I trial evaluationof Her2Bi armed ATC infusions with specific focus ona CRPC cohort to review safety and preliminary efficacy.Evaluation of immune responses in our phase I clinical trialin CRPC patients suggests that infusions of Her2Bi-armedATC (aATC) were safe and induced antitumor responses.Our findings suggest that Her2Bi-armed ATC therapy maybe an effective, nontoxic, tumor-specific treatment for Her2-positive CRPC.

2. Patients and Methods

The study was a phase I trial conducted at Roger WilliamsHospital, Providence, Rhode Island. The study and consentwere approved by the local institutional review board. Theprimary objective of the study was to perform a phase Idose-escalation using Her2Bi armed ATC in prostate cancerpatientswith 0–3+Her2 positivity.The cohort of patientswithmetastatic CRPC were treated under an FDA approved INDsponsored by LGL.

2.1. Eligibility for Prostate Cancer Cohort. Castration resistantpatients with pathologically documented adenocarcinoma ofthe prostate that progressed despite androgen suppressiontherapy irrespective of their HER-2/neu expression statuswere eligible. Progression was determined by at least one ofthe following: rising PSA, increase in measurable disease,or new areas of bone metastases. Patients were required tohave measurable or evaluable disease and at least 4 weeksof rest after chemotherapy or radiation before enrollmentinto the protocol. Her2 staining was not performed since itwas not standard of care. Concurrent radiation treatmentwas not permitted; however, local irradiation of metastaticdisease was allowed for local pain control and/or functionalimpairment due to localized lesions.

Cell infusions could begin as early as 1 week after comple-tion of local irradiation if the toxicity had resolved based onthe assessment of the treating physician. Karnofsky perfor-mance score ≥ 60% or ECOG score 0–2 was required, withminimum life expectancy of 3 months. Hormone therapy(except LHRH analogue) had to be discontinued at least fourweeks prior to the initiation of armed-ATC infusions. Eachpatient had to sign a written informed consent to treatmentafter being informed of alternatives, potential benefits, sideeffects, and risks. No history of other malignancies waspermitted unless it was in situ squamous cell carcinoma orbasal cell carcinoma of the skin, or other cancers in remissionfor 5 years or more.

Exclusion criteria included history of myocardial infarc-tion in last 12 months, impaired left ventricular function(LVEF ≤ 45% by MUGA), congestive heart failure, un-controlled hypertension, or significant pulmonary disease(DLCO >60%) on pulmonary function tests. Normal bonemarrow and renal and liver function were required. Patientswith conditions or medications leading to immunosuppres-sion were excluded.

2.2. Production of Heteroconjugated Bispecific Antibody(Her2Bi). Anti-CD3monoclonal antibody (OKT3, CentocorOrtho-Biotech, Raritan, NJ) was heteroconjugated to anti-Her2 monoclonal antibody (Herceptin, Genentech, SouthSan Francisco, CA) to produce the anti-OKT3 x anti-Her2bispecific antibody (Her2Bi) as previously described [24].

2.3. Leukopheresis, TCell Expansion, andArmingwithHer2Bi.peripheral blood mononuclear cells (PBMC) were collectedto obtain lymphocytes for ATC expansion from 1 or 2leukopheresis. PBMCwere activated with 20 ng/mL of OKT3and expanded in 100 IU/mLof IL-2 to generate 40–320 billionATC during a maximum of 14 days of culture under cGMPconditions as described [15, 16]. The cells were grown inbreathable flasks (FEPBagType 750-C1, American FluorosealCorporation, Gaithersburg, MD) in RPMI 1640 medium(Lonza) supplemented with 2% pooled heat inactivatedhuman serum (Valley Biomedical, Winchester, VA). ATCwere split approximately every 2-3 days based on cell counts.After 14 days of culture, ATC were harvested and armedwith a pretitrated dose of 50 ng of Her2Bi/106 ATC, washed,and cryopreserved in multiple aliquots. Aliquots of each bagwere sent for bacterial and fungal cultures (Roger Williams

Prostate Cancer 3

Treatment schema for prostate cancerSc

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aATC infusionsGM-CSF 250𝜇g/m2/dose

IL-2 300,000 IU/m2/day

Figure 1: The treatment schema. Her2Bi armed ATC (aATC) wereadministered twice weekly for four consecutive weeks. All patientsreceived subcutaneous IL-2 (300,000 IU/m2/day) and GM-CSF(250 𝜇g/m2/twice weekly), beginning 3 days before the first aATCinfusion and ending 1 week after the last aATC infusion. Immunetesting was performed at indicated time points.

Medical Center Pathology Laboratories), endotoxin testing(Lonza, Inc., Walkersville MD), and mycoplasma testing(Bionique Testing Laboratories, Inc., Saranac Lake, NY).No exogenous IL-2, OKT3, or other culture reagents (e.g.,medium components) are present in the final cryopreservedproduct.Thephenotype viability, proliferation, and responsesof ATC to IL-2 did not change after cryopreservation. Thecytotoxicity mediated by ATC armed prior to cryopreser-vation was equivalent to those armed after thawing ofcryopreserved ATC.

2.4. Infusions of Her2Bi Armed ATC. T cells were infusedonly after standard operating procedures for identifying thecryopreserved product and the patient at the bedside. Theinfusion rate was calculated based on an infusing rate ofno more than 5 EU of endotoxin/kg/hour. Armed ATC weregiven over 5–15mins with monitoring of vital signs beforeand every 15mins up to 1 hr after infusion. All vital signs andside effects were recorded on the patient’s chart using theNCIimmunotherapy toxicity table. Patients were observed up to 6hours after their infusions. The dose levels for each infusionwere 2.5, 5, 10, and 20 billion. Each patient would receive atotal of 8 doses of armed ATC given twice weekly for 4 weeks.If the patients encountered toxicities related to armed ATC,modifications of the dose or holding the administration wasrequired. The patients also received subcutaneous injectionsof interleukin-2 [IL-2] (3.0 × 105 IU/m2/day) starting 3 daysbefore the 1st armed ATC infusion and ending 7 days afterthe last armed ATC infusion. GM-CSF (250𝜇g/m2 twice perweek) was administered starting 3 days before the first armedATC infusion and ending 7 days after the last dose of armedATC (Figure 1).

2.5. Study Design and Statistical Plan. Three patients wereentered at each dose level (Figure 1). Escalation of the

dose was based on “cell-based” toxicity defined as toxicitiesattributed directly to the infusion of armed ATC. Doseescalation was done if more than 80% of planned-armedATC dose was delivered without any grade 3 or greatertoxicity attributed to therapy. The cohort was expanded tosix patients at any particular dose level if one of the firstthree patients had their infusions stopped due to grade 3 orgreater nonhematological toxicity or received less than 80%of planned dose of armed ATC. If, in the expanded cohort,one additional patient at this dose level had the infusionsdiscontinued due to toxicities or had less than 80% of theplanned dose delivered, then this particular dose level was tobe defined as the one at which dose limiting toxicity (DLT)had occurred and further dose escalation was terminated.The maximum tolerated dose (MTD) would be defined asthe dose level below the one at which dose limiting toxicityoccurred and this would be the recommended phase II dose.The phase I trial consisted of metastatic breast cancer andprostate cancer patients. This is a report only on the prostatecancer patients enrolled on this study. Response was assessedby monitoring PSA every 4 weeks until progression. Due tosmall sample size, only descriptive clinical parameters of PSAdecline, time to progression, and overall survival are used.Immunologic markers were assessed on the patients whencell numbers were permitted.

2.6. Immunologic Monitoring. The number of IFN𝛾 produc-ing cells from fresh PBMC frommenbefore and at the varioustime points after infusions of armed ATC was detectedby IFN𝛾 specific EliSpot assay (BD Biosciences, San Jose,CA). Spots were captured and counted on CTL Immunospotcounter using Immunospot software version 4 (CellularTechnology Ltd, ShakerHeights, OH).We comparedwhetherin vivo lysis of tumors by the armed ATC will inducememory T cells capable of inducing apoptosis when exposedin vitro to tumor cells (a recall response) and to look fordifferences in the precursor frequency [25–29].Mean changesin the induction of IFN𝛾 EliSpots responses before and afterimmunotherapy were plotted over time to explore how longthey can remain elevated. Paired 𝑡-test or Wilcoxon signedrank testwas used in these comparisons.Themeans,medians,and standard deviations were calculated for the number ofIFN𝛾 EliSpots.

2.7. Endogenous Cytokine Production. In order to deter-mine whether multiple infusions of armed ATC induced animmune response, Th

1and Th

2cytokines were evaluated in

the serum of the patients using a 25-plex human cytokineLuminex Array (Invitrogen, Carlsbad, CA) on a Bio-Plexsystem (Bio-Rad Lab., Hercules, CA). Data was analyzed asfunction of number of infusions [25–29] and the in vivoresponse was calculated as the mean Th

1[IL-2+IFN𝛾]/Th

2

[IL-4+IL-5] ratio of cytokines.

3. Results

3.1. Patient Characteristics. Here we report pilot data on 8CRPC patients from our phase I clinical trial; the remaining

4 Prostate Cancer

Table 1: Patient characteristics, prior therapy, disease status, and toxicity.

FG # Age/PS Gleasonscore

aATC doselevel

Metastatic diseasesites Prior therapy Disease status OS in

daysGrade 3toxicity

1 00469 66/PS-2 7 20 billion Bone, lymph nodes,lung, and liver

Hormonesdocetaxel

+ estramustine

Progressed duringaATC infusions 67 None

2 00488 82/PS-2 9 20 billion Bone Hormonesmitoxantrone

Progressed duringaATC infusions 143 None

3 00566 75/PS-1 8 20 billion Bone Hormones Progressed duringaATC infusions 699 Chills

4 00594 85/PS-1 8 20 billion Lymph nodes, bone Hormones Progressed duringaATC infusions 136 Chills

5 60163 76/PS-1 6 40 billion Bone, lung Hormones PR 598 Chills6 60202 80/PS-1 8 40 billion Bone Hormones MR 294 Chills7 91760 69/PS-1 6 80 billion Bone, lung Hormones MR 550 Chills, MalaisePS: performance status; PR: partial remission;MR:minor response; OS: overall survival.Therewere no grades for toxicities on theNCI-immunotherapy toxicityprotocol.

Table 2: Phenotype of harvest product.

FG # Total harvest ×109 % viability %CD3 harvest %CD4 harvest %CD8 harvest1 35 95 89 58 342 38 95 93 75 223 48.6 91 99 50 504 65.4 87 79 19 605 139 94 89 61 356 171 87 90 78 16

22 metastatic breast cancer patients will be reported else-where. One of the enrolled CRPC patients did not receivetherapy due to rapid progression of his disease. The medianage of the 7 patients with metastatic CRPC treated on studywas 76 years (range 66–85 years). Prior therapy includedLHRH analogue therapy or antiandrogen therapy for allpatients. One patient had received prior docetaxel-basedchemotherapy, one patient had received prior mitoxantronechemotherapy, and one patient had multiple hormonalmaneuvers consisting of ketoconazole, steroids, and estrogensequentially (Table 1).

3.2. ATC Characteristics. Patients received a total of 8 dosesof 2.5, 5, 10, and 20 billion armed ATC given twice weekly for4 weeks. Characteristics of ATC are shown in Table 2.

3.3. Clinical Responses, PSA Levels, and Toxicities. There wasa decrease in narcotic use in 2 of the 7 men possibly dueto decreased bone pain. In addition to reduction of medi-cation for bone pain associated with metastases, two CRPCpatients with bulky disease infused with 40 (60202) and 80(91760) billion Her2Bi-armed ATC, respectively, had minorresponses observed as transient decreases in PSA levels (892to 767 ng/mL and 1140 to 1046 ng/mL, resp.) that had short-term persistence after the last cell infusion (Figure 2(a)). Athird CRPCpatient (60163), who received 40 billion cells, hada partial responsewithin 6months of completing therapywiththe patient’s PSA levels decreasing by >50% (Figure 2(b)).

Two patients showed an increase in their PSA levels aftercompletion of 8 infusions of Her2Bi armed ATC comparedto their preIT baseline levels (Figure 2(c)). Therapy was welltolerated and all patients received at least 80% of the planneddose.Thedoses administered,OS outcomes, and toxicities areincluded inTable 1.Our data showencouragingOS for a smallcohort of men with HRPC.

3.4. Induction of Interferon Gamma (IFN𝛾) EliSpots. FreshPBMC were stimulated with PC-3 cells to induce IFN𝛾EliSpots. Figures 3(a), 3(b), and 3(c) show the increases inIFN𝛾 expressing cells after armed ATC infusions. There wasa noticeable increase in the number of IFN𝛾 EliSpots atpostinfusion #5 compared to baseline (prior to armed ATCinfusions [preIT]) in two patients (FG60163 and FG60202)and postinfusion #1 in third patient (FG91760). These datashow that either memory helper T cells or cytotoxic T cellswere induced by Her2Bi armed ATC infusions, preexist inthe circulating peripheral blood lymphocytes, and respondimmediately to direct stimulation with prostate tumor anti-gens on PC-3 targets.

3.5. Infusions of Armed ATC Induce Endogenous CytokineProduction. The in vivo response calculated as the meanTh1[IL-2+IFN𝛾]/Th

2[IL-4+IL-5] ratio of cytokines remained

predominantly Th1polarized throughout treatment. Figures

4(a)–4(c) show Th1and Th

2cytokines patterns for patients

FG60163, FG60202, and FG91760, respectively. All three

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Figure 2: (a)The transient decrease in PSA levels in two CRPC patients (60202 and 91760) who hadminor responses. (b) A partial responder(60163) with the PSA levels declining by >50% within 6 months of completing therapy. (c) PostIT PSA levels in two nonresponding patients(FG00594 and FG00566) compared to their preIT baseline levels.

patients (2-MR and 1-PR) showed increases in IL-2, GM-CSF, and IFN-𝛾 (Th

1cytokines) compared to baseline levels

(preIT) in sequential testing of the serum. Th2cytokine IL-

10 also showed comparable pattern as Th1cytokines. Other

Th2cytokines IL-4 and IL-5 could not be detected. These

findings are consistent with increased specific cytotoxic Tlymphocyte responses observed in EliSpots of patient postin-fusion PBMC exposed to tumor cells. These observationsshow that the endogenous immune systems of cancer patientscan be shifted to favor an antitumor immune environment byinfusion of armed ATC.

4. Discussion

Our phase I study treating metastatic CRPC patients withHer2Bi-aATC shows that armed ATC infusions are safe andwell tolerated leading to 2 minor and 1 partial responses inCRPC patients who received doses of 40 and 80 × 109 aATC,

respectively. The PSA levels decreased in 3 of 7 patients andone of the 7 patients had a >50% decline in PSA below thebaseline (PR). In two patients who were studied for IFN-𝛾EliSpots and cytokine response, there was a marked increasein IFN-𝛾 EliSpots and serum Th

1cytokines (IL-2, GM-CSF,

and IFN-𝛾) after the 5th infusion (Inf #5) over baseline whenno increases in Th

2cytokines were observed.

Armed ATC not only may function as Her2 specificcytotoxic T lymphocytes in prostate cancer patients, but alsomay divide and secrete Th

1cytokines and RANTES and

MIP1-𝛼 after binding toHer2+ tumors leading to recruitmentof endogenous T cells and monocytes leading to in situvaccination at the tumor site [30]. Tumor lysis mediated bytargeting T cells together with release of IL-2, IFN-𝛾, andGM-CSF may provide the milieu to induce immunizationof endogenous immune cells to tumor associated antigens(TAA). Therefore, multiple rounds of armed ATC infusionscould then eventually induce systemic immune responses

6 Prostate Cancer

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Figure 3: Number of IFN-𝛾 producing cells when incubated overnight with prostate cancer cell line PC-3 in PBMC collected at baseline(preimmunotherapy [preIT]), during infusions (post inf#), and postimmunotherapy (1 week postimmunotherapy [1W postIT], 1 monthpostimmunotherapy [1M postIT], and one year postimmunotherapy [1Y postIT]). (a) Increased number of IFN-𝛾 producing cells duringinfusions and postIT time points in a partial responder (FG60163) when PBMC were incubated with prostate cancer specific PC-3 targets.(b) and (c) Enhanced IFN-𝛾 responses in minor responders (FG60202 and FG91760).

that could be detected as IFN-𝛾 EliSpots in the peripheralblood mononuclear cells (PBMC) from patients.

Recently, Sipuleucel-T/APC8015/Provenge showed a re-lapse free survival benefit in a phase III placebo controlledtrial [1]. For this study, PBMCwere collected and pulsed withprostatic acid phosphatase antigen and GM-CSF.The antigenloaded dendritic cell enriched preparation was then adminis-tered to patients. Final results of the placebo controlled trialinvolving 127 patients revealed an improvement in OS at 3years. OS was 11% in the placebo group and OS was 33%in the Sipuleucel treated metastatic prostate cancer cohort(𝑃 = 0.02).The results of a larger phase III placebo controlledtrial [IMPACT1] in asymptomatic metastatic CRPC patientsshowed an OS benefit with the therapy, but no PFS benefit.The agent was well tolerated and, due to the overall survivalbenefit, the FDA approved Sipuleucel-T as the first cell-basedimmunotherapy approved by the FDA for any cancer.

Other immunotherapy approaches have been tested inpatients with metastatic CRPC. Initial studies with GVAX,

an allogeneic cellular therapy consisting of a combinationof two prostate carcinoma cell lines (PC-3 and LnCAP)with a modified GM-CSF gene, appeared to be promising.In phase II trials, GVAX was well tolerated and showedclinical promise with a median survival of 26 months andmedian time to progression of 5months in patients with bonemetastases [31]. However, a phase III trial of single agentGVAX versus docetaxel in asymptomatic CRPC was closedearly due to a disappointing interim analysis, and a phase IIItrial of docetaxel +/− GVAX in symptomatic CRPC showedan increased number of deaths [24]. Recently, a PSA directedtherapeutic vaccine (ProstVacVF) revealed a 44% reductionin death rate favoring the vaccine arm over placebo controlafter 13 years of follow-up [32]. There was no change in PFSwhen the primary endpoint of PFS was reported in 2004, butafter prolonged follow-up, a statistically significantOS benefitwas noted.

There is encouraging preliminary evidence to sup-port investigations targeting CRPC with aATC. Multiple

Prostate Cancer 7

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IFN-𝛾

(c)

Figure 4: Profile of serum cytokines. Analysis of sequential serum samples at baseline (preimmunotherapy [preIT]), during infusions(post inf#), and postimmunotherapy (1 week postimmunotherapy [1W postIT], 1 month postimmunotherapy [1M postIT], and one yearpostimmunotherapy [1Y postIT]) shows increased levels of IL-2, IFN-𝛾, GM-CSF, and IL-10 and meanratio of Th

1/Th2= [IL-2+IFN𝛾]/[IL-

4+IL-10] shows a dominant Th1type response during aATC infusions in partial responder CRPC patient (FG60163). (b) and (c) Similar

cytokine profiles and Th1cytokine responses were seen in two minor responder CRPC patients (FG60202 and FG91760) during and after

aATC infusions.

investigators have used infusions of BiAb to activate T cellsin vivo and enhance targeting and killing of Her2+ tumors[33–38]. In a phase II advanced prostate and renal cancertrial using the BiAb MDX-H210 (15𝜇g/m2) IV and GM-CSF5 𝜇g/kg/day subcutaneously for 4 days repeated weekly for6 weeks, 7 of 20 (35%) patients with prostate cancer hadobjective responses (>50% decline in PSA) [38].

There are a number of innovative and novel componentsof this approach. As described earlier, aATC can even targetbreast cancer cells lines [24] and prostate cancer cell lines(PC-3, LNCap) [23] with low HER2 receptor expression. Theability to target tumors that express low numbers of Her2receptors is rationale for including all patients regardlessof their Her2 expression. This study shows that multipleinfusions of aATC induced immune responses that couldbe detected by reexposure to prostate cancer antigens inIFN-𝛾 EliSpot assays. These data suggested that the armedATC infusions “vaccinated” the patients against a wholearray (unknown) of their own prostate cancer antigens andgenerated anti-tumor CTL or T cell helper activity. In anearlier study, we showed that in women with metastaticbreast cancer low dose IL-2, low dose GM-CSF, and eightHer2Bi armedATC infusions induced cytotoxic T cell activitydirected at SK-BR-3 breast cancer cell targets that couldbe detected up to 4 months or more after immunotherapy

[30]. The results from these studies suggest that this novelcombination of antibody targeting and non-MHC restrictedT cell-mediated cytotoxicity may have high clinical impactand therapeutic benefit in metastatic CRPC patients.

5. Conclusions

This phase I dose escalation study showed that the treatmentof metastatic CRPC patients with Her2Bi-aATC was safe andwell tolerated. CRPC patients who received 40 and 80 × 109aATC doses showed 1 partial and 2 minor responses, as wellas induced antitumor immune responses. These data providea strong rationale for developing phase II clinical trials todetermine whether aATC are effective for treating CRPC.

Conflict of Interests

Lawrence G. Lum is cofounder of Transtarget Inc. UlkaVaishampayan, Archana Thakur, Ritesh Rathore, and NicolaKouttab declare that they have no competing interests.

Authors’ Contribution

Ulka Vaishampayan and Archana Thakur wrote the paper.Lawrence G. Lum, Ritesh Rathore, Nicholas Koutab were

Prostate Cancer 9

involved in the design, regulatory submissions, writing ofthe protocol, clinical management of the protocol, and cellinfusions. Lawrence G. Lum, Ritesh Rathore, and NicholasKoutab were involved in producing the cGMP product,immune evaluation assays, and data management and collec-tion. Archana Thakur and Lawrence G. Lum were involvedin data analysis. Ulka Vaishampayan, Archana Thakur, andLawrence G. Lum contributed equally to this paper.

Acknowledgments

The authors acknowledge the efforts of technical (JanelleSmith, Pam Davol, and Estie Palushock) and clinical coor-dinating staff (Wendy Young, Annette Olson, and Lori Hall)who took a special interest in coordinating patient care andEileen Silveira who provided nursing support for the clinicalinfusions. Lawrence G. Lum was supported in part by grantsfrom the Leukemia and Lymphoma Society (TRP 6066-06),the National Cancer Institute (R01 CA 092344), and startupfunds from Cancer Center of Roger Williams Hospital.

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